Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

• the invention relates to novel benzimidazole derivatives, their preparation and their use for the preparation of medicaments for the treatment and prophylaxis of diseases associated with microglial activation, and of T-cell-mediated immunological diseases, as well as pharmaceutical preparations containing the novel benzimidazole derivatives contain.
• the immune system includes a variety of cells and tissue complexes that communicate primarily via soluble factors. It is known that many immunological diseases are due to an imbalance of
• Interleukin 12 in the pathogenesis of autoimmune diseases Special mention should be made of diseases characterized by a T-cell-mediated inflammatory reaction, such as multiple sclerosis, diabetes, inflammatory bowel diseases (chronic inflammatory bowel diseases).
• diseases characterized by a T-cell-mediated inflammatory reaction such as multiple sclerosis, diabetes, inflammatory bowel diseases (chronic inflammatory bowel diseases).
• cytokine interleukin 12 is produced by phagocytic cells, such as
• Macrophages / monocytes, dendrites, B cells, and other antigen presenting cells (APCs) produce and affect both natural killer (NK) and T lymphocyte functions.
• IL 12 can stimulate the production of interferon gamma (IFN ⁇ ) in both cell types.
• IFN ⁇ interferon gamma
• T-lymphocytes can be broadly divided into two categories which are characterized by the expression of certain surface antigens (CD4 and CD8): CD4-positive T-cells (helper T-cells) and CD8-positive T-cells ( cytotoxic T cells).
• CD4 and CD8 CD4-positive T-cells
• the CD4 cells can turn into T-helper Cells 1 (Th1) and T helper cells 2 (Th2).
• the Th1-mediated immune responses are associated with the pathogenesis of numerous immune diseases, especially the autoimmune diseases, such as: type I insulin-dependent diabetes mellitus (IDDM), multiple sclerosis, allergic contact dermatitis, psoriasis, rheumatoid arthritis, chronic inflammatory bowel disease ( "Inflammatory bowel diseases” - Crohn's disease, ulcerative colitis), lupus diseases and other collagenoses as well as acute rejection reactions in the allograft ("host-versus-graft” graft-versus-host disease).
• IDDM type I insulin-dependent diabetes mellitus
• multiple sclerosis sclerosis
• allergic contact dermatitis psoriasis
• rheumatoid arthritis chronic inflammatory bowel disease ( "Inflammatory bowel diseases” - Crohn's disease, ulcerative colitis)
• lupus diseases and other collagenoses as well as acute rejection reactions in the allograft ("host-versus-graft” graft-versus-
• Interleukin 12 is known to play a critical role in the regulation of the Th1 response. Interleukin 12 induces in these cells the production of mainly IL-2, IFN ⁇ , TNF ⁇ and TNF ⁇ (Mosmann and Sad, Immunol., Today 17: 138-146 (1996); Gately et al., Annu., Rev. Immunol., 16: 495) - 521 (1998)). Specifically, IFN ⁇ is a potent mediator of IL-12 action.
• interferon gamma may be responsible, for example, for the MHC II (major histocompatibility complex) -associated autoimmune diseases (In addition, there is also sufficient evidence regarding a pathological role of interferon gamma in allergic diseases as well as sarcoidosis and psoriasis. , Immunol., 62: 61-130 (1996); Basham et al., J.
• IL-12 and IL-12 / IL-18-induced IFN ⁇ from NK cells are essential in the pathomechanism of non-T cell-mediated inflammatory responses (eg, "Toxic shock Syndromes ", endotoxemia, sepsis and septic shock, ARDS," first dose response "in antibody therapy eg OKT3 administration in allograft) (Kum et al., Infect Immun. 69: 7544-7549 (2001); Arad et al.
• IL-12 also plays a role in inflammation with currently unclear pathomechanisms (eg eclampsia) (Hayakawa et al., J. Reprod Immunol 47: 121-138 (2000); Daniel et al., Am J. Reprod Immunol 39: 376-380 (1998)).
• TNF ⁇ inflammatory mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated inflammatory reactions, such as TNF ⁇ .
• infectious diseases such as mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated mediated
• Activation of the microglia is a central step in the inflammatory process of almost all degenerative diseases of the central nervous system.
• the microglia may remain in the activated state for a prolonged period of time by causing various inflammatory factors, e.g. Reactive oxygen / nitrogen intermediates, proteases, cytokines, complement factors and neurotoxins, produce and secrete. These in turn cause neuronal dysfunction and
• microglia The activation of microglia can be done by various stimuli, such as. B. A ⁇ peptide ( ⁇ -amyloid, Araujo, DM and Cotman, CM., Brain Res. 569: 141-145 (1992)), prion protein, cytokines, or by cell fragments (Combs, CK et al., J. Biol. Neurosci., 19: 928-939 (1999); Wood, P.L., Neuroinflammation: Mechanisms and Management, Humana Press (1998).
• a ⁇ peptide ⁇ -amyloid, Araujo, DM and Cotman, CM., Brain Res. 569: 141-145 (1992)
• prion protein prion protein
• cytokines cytokines
• cell fragments Combs, CK et al., J. Biol. Neurosci., 19: 928-939 (1999); Wood, P.L., Neuroinflammation: Mechanisms and Management, Humana Press
• Benzimidazoles which inhibit activation of the microglia after stimulation with the A ⁇ peptide are described in WO 01/51473. That's it, too known to inhibit microglia activation benzimidazoles for the treatment of neuroinflammatory diseases such as AIDS dementia, Amyotrophic Lateral Sclerosis, Creutzfeldt-Jakob Disease, Down's Syndrome, diffuse Lewy Body Disease, Huntington's Disease, Leukencephalopathy, Multiple Sclerosis, Parkinson's disease, Pick's disease, Alzheimer's disease, stroke, temporary lobe epilepsy and tumors.
• neuroinflammatory diseases such as AIDS dementia, Amyotrophic Lateral Sclerosis, Creutzfeldt-Jakob Disease, Down's Syndrome, diffuse Lewy Body Disease, Huntington's Disease, Leukencephalopathy, Multiple Sclerosis, Parkinson's disease, Pick's disease, Alzheimer's disease, stroke, temporary lobe epilepsy and tumors.
• EP 0 104 727 A1 describes benzimidazole derivatives which are unsubstituted in the 1-position and have an alkyl group in the 2-position.
• Substituents on the benzene ring of the derivatives are i.a. Pyridyloxy, pyridylalkyl, pyridylalkyloxy and pyridyloxyalkanediyl radicals.
• Phenyl or heteroaryl radical and on the fused benzene ring u.a. at least substituted with a substituted alkoxy, alkylamino, alkylsulfonyl and alkylsulfoxide radical. It is stated that these substances can be used as an active ingredient in pharmaceutical preparations for a variety of potential indications.
• US-A-5,552,426 discloses substituted benzimidazoles which are in the 1-position and the like. a phenyl or naphthyl radical and in the 2-position u.a. have a phenyl or heterocycle radical.
• the fused benzene ring of the benzimidazoles is preferably substituted with an alkoxy or aminoalkoxy radical. Such compounds are said to be efficacious against diseases based on ⁇ -amyloid peptide-associated neurotoxicity.
• Benzimidazolderivate be given as active ingredients which in 1-position, inter alia, with a phenyl or substituted phenyl radical and in the 2-position with an alkoxy radical are substituted.
• Substituents on the benzene ring of the active compounds may include alkyl, nitro, halogeno, alkoxy, amino, ester, amide, alkanediylalkoxy and alkanediylamino radicals.
• EP 0 520 200 A2 discloses benzimidazole derivatives which have aryl radicals substituted in the 1 position and mono-, di-substituted or unsubstituted amino radicals in the 2-position.
• the benzene ring of the benzimidazole skeleton may be substituted with halogen, trifluoromethyl and / or cyano. These compounds are used to treat diseases associated with increased activation of Ca channels.
• benzimidazole derivatives are also indicated, which are used for the treatment of cystitis. These compounds may be in 1-position and the like. Having phenyl, naphthyl and unsaturated heterocycle radicals. In the 2-position, the derivatives may be substituted with alkoxy, phenylalkoxy, naphthylalkoxy, heterocyclicalkoxy or unsaturated heterocyclicalkoxy radicals.
• the benzene ring of the backbone of the derivatives can be reacted with nitro, alkanoyl, amino, alkyl, alkoxy, cycloalkyl, heterocycle, unsaturated heterocycle, halogeno, alkylthio, hydroxyalkylidenyl, hydroxyalkylidenylamino, aminoalkylidenyl, Substituted aminoalkoxy, hydroxyalkyl, Heterocyclusalkoxy-, Aminoalkylidenyl- or trifluoromethyl radicals.
• EP 0 531 883 A1 discloses condensed 5-membered heterocycles, for example substituted benzimidazole derivatives, these compounds according to the general description of the compounds in the 1-position preferably having a substituted alkyl radical and in the 2-position for example with an O-alkanediyl, S Alkandiyl, NH-alkanediyl, N (alkyl) alkanediyl, SO-alkanediyl or S0 2 -Alkandiylrest substituted.
• the compounds described are said to have antithrombic activity.
• non-steroidal anti-inflammatory drugs COX II inhibitors
• COX II inhibitors COX II inhibitors
• novel benzimidazole derivatives according to claim 1 further by the use of a benzimidazole derivative according to the invention for the preparation of a medicament for interrupting IL 12 and INF Y production in cells of monocytic origin or T cells and NK cells.
• microglial inhibitors are useful in the treatment of many diseases caused by the increased production of cytokines such as TNF ⁇ . ⁇ , IFN ⁇ , IL-2 and IL12 are triggered, such as inflammatory diseases that are not based on neuroinflammation, Autoimmune diseases, allergic and infectious diseases, toxin-induced inflammation, pharmacologically induced inflammatory reactions and pathophysiologically relevant inflammatory reactions of currently unclear origin.
• benzimidazole derivatives according to the invention have the following general structural formula I:
• R 1 is a phenyl group optionally containing up to three of the following
• R 3 is H, OH or 0-C 1-6 -alkyl
• R 5 and R 5 each independently represent a radical selected from the group consisting of C ⁇ -6 alkyl, C 2- 6 alkenyl, C 2- 6 -alkynyl, where one C atom to O, S, SO, S0 2 , NH, NC 1-3 alkyl or N-C ⁇ -3 -Akaloyl can be exchanged, furthermore (C 0-3 alkanediyl-C 3-7 - cycloalkyl), wherein in a five-membered cycloalkyl ring, a ring member ring-N or ring -0 may be and in one six- or seven-membered cycloalkyl ring or two ring members can be ring N and / or ring O atoms, respectively, where the ring N atoms optionally substituted by C ⁇ -3 alkyl or C ⁇ -3 - Alkanoyl may be substituted, as well as (C 0-3 -alkanediyl-phenyl)
• R 1 is a phenyl group optionally substituted with up to two of the following substituents independently of one another: F, Cl, OH, OR 4 , OCOR 4
• R 4 or two substituents on R 1 benahbarte form a -0-CH 2 -0- or - CH 2 -CH 2 -CH 2 group.
• R 2 is a monocyclic 5-6 membered heteroaryl group containing 1-2
• R J is H.
• R * and R 4 ' are independently of one another C 2 -2- perfluoroalkyl, C 1 -alkyl.
• benzimidazole derivatives in which R and R 5 'independently of each other C ⁇ - 6 alkyl, wherein a carbon atom may be replaced by O, S, SO, SO 2 , C 3-5 cycloalkyl-Co-3
• N or an O may be, wherein the ring nitrogen is optionally substituted by C- ⁇ -3 alkyl or C 1-3 alkanoyl,
• NH2 NH C ⁇ -3 alkyl, NH C ⁇ -3 alkanoyl, N (C 1-3 alkyl) 2, N (C ⁇ -3 alkyl) (C 1-3 alkanoyl), COOH, CONH 2 and all heteroaryl aforementioned with one or two substituents from the group consisting of F, Cl, CH 3 , C 2 H 5 , OCH 3 , OC 2 H 5 , CF 3 , C 2 F 5 may be substituted,
• R 5 and R 5 together with the nitrogen atom form a 5-7 membered heterocycle which may contain a further oxygen, nitrogen or sulfur atom and may be substituted by C__
• A is straight chain C 3-6 alkylene.
• B is COOH or CONH 2 each attached to a carbon atom of group A.
• Particularly preferred are the following benzimidazoles: 6 - [[1- (4-methylphenyl) -2- (3-pyridinyl) -1 / - / - benzimidazol-6-yl] oxy] hexanoic acid 5 - [[1- (4- Methylphenyl) -2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] pentanoic acid 4 - [[1- (4-methylphenyl) -2- (3-pyridinyl) -1 / - / - benzimidazole] 6-yl] oxy] butyric acid 6 - [[1- (4-methylphenyl) -2- (4-pyhdinyl) -1 / - / - benzimidazol-6-yl] oxy] hexanoic acid 6
• the present invention comprises the physiologically tolerated salts of the abovementioned compounds, in particular the acid salts of the nitrogen bases of the benzimidazole derivatives according to the invention, and also the salts of carboxylic acids of the derivatives according to the invention with bases.
• the benzimidazole derivatives of the invention may have one or more asymmetric centers, so that the compounds may occur in several isomeric forms.
• the compounds of the formula I can also be present as tautomers, stereoisomers or geometric isomers.
• the invention also includes all possible isomers, such as E and Z isomers, S and R enantiomers, diastereomers, racemates and mixtures thereof, including the tautomeric compounds. All these isomeric compounds are - even if not expressly stated - part of the present invention.
• the isomer mixtures can be separated into the enantiomers or E / Z isomers by customary methods, such as, for example, crystallization, chromatography or salt formation.
• heteroaryl groups present in the benzimidazole compounds according to the invention are composed of five or ten skeletal atoms and may contain one or two heteroatoms. Heteroatoms are oxygen (O), nitrogen (N) and sulfur (S). Examples of monocyclic heteroaryl groups are pyrrolyl, thienyl, furanyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl.
• bicyclic heteroaryl group examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, benzimidazolyl, indazolyl, quinolyl, isoquinolyl, phthalazinyl, quinazolinyl, cinnolinyl, quinoxalinyl, naphthyridinyl.
• the heteroaryl group is part of R 1 , the bond to the N of the benzimidazole occurs via a carbon atom.
• Heteroaryl radicals can be bonded in any manner to the benzimidazole skeleton or another group, for example as 2-, 3- or 4-pyridinyl, as 2- or 3-thienyl or as 1-imidazolyl.
• Alkyl groups can be straight-chain or branched. Examples of alkyl groups are methyl, ethyl, n-propyl, so-propyl, n-butyl, sec-butyl, etf-butyl, n-pentyl, sec-pentyl, f-pentyl, neo-pentyl, n-hexyl , se / -Hexyl.
• Perfluorinated alkyls are preferably CF 3 and C 2 F 5 .
• Cycloalkyl groups are in each case preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.
• saturated heterocyclic ring or cycloalkyl having one or more heteroatoms are: piperidine, pyrrolidine, tetrahydrofuran, morpholine, piperazine, hexahydroazepine and 2,6-dimethylmorpholine, N-phenylpiperazine, 2-methoxymethyl-pyrrolidine , Piperidine-4-carboxamide, thiomorpholine, thiazolidine, wherein the linkage can take place via optionally present ring N atoms.
• straight-chain or branched alkylene for A having up to six C atoms there may be mentioned, for example: ethylene, propylene, butylene, pentylene, hexylene, furthermore 1-methylethylene, 1-ethylethylene, 1-methylpropylene, 2-methylpropylene, 1-methyl- butylene, 2-methylbutylene, 1-ethylbutylene, 2-ethylbutylene, 1-methylpentylene, 2-
• Methylpentylene, 3-methylpentylene and analogous compounds are Methylpentylene, 3-methylpentylene and analogous compounds.
• A may be disubstituted, preferably monosubstituted, with OH, NH 2, NH-d -3 - alkyl or NH-C ⁇ -3 alkanoyl.
• the physiologically acceptable acid salts of the nitrogen bases of the benzimidazole derivatives according to the invention can be formed with inorganic and organic acids, for example with oxalic acid, lactic acid, citric acid, fumaric acid, acetic acid, maleic acid, tartaric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, sulfuric acid, p-toluenesulfonic acid and methanesulfonic acid.
• the inorganic or organic bases are also suitable, which are known for the formation of physiologically acceptable salts, such as alkali metal hydroxides, in particular sodium and potassium hydroxide, alkaline earth hydroxides, such as calcium hydroxide, ammonia, and amines, such as ethanolamine, Diethanolamine, triethanolamine, N-methylglucamine and tris (hydroxymethyl) methylamine.
• physiologically acceptable salts such as alkali metal hydroxides, in particular sodium and potassium hydroxide, alkaline earth hydroxides, such as calcium hydroxide, ammonia, and amines, such as ethanolamine, Diethanolamine, triethanolamine, N-methylglucamine and tris (hydroxymethyl) methylamine.
• the compounds of formula I inhibit the activation of microglia and the production of interleukin 12 (IL 12) and interferon ⁇ (IFN ⁇ ).
• the invention therefore also relates to the use of a compound of formula I, and their optical or geometric isomers or their tautomers or physiologically acceptable salt for the manufacture of a medicament for the treatment or prevention of a microglial activation associated disease, in particular by overproduction of IL 12 and IFN ⁇ -induced disease and induction of interleukin 10 (IL-10).
• the compounds according to the invention are used for the treatment of a T-cell-mediated, specifically Th-1-cell-mediated, immunological disease and of non-T-cell-mediated inflammatory reactions.
• the compounds of the invention are used for the preparation of a medicament for interrupting IL 12 and INF ⁇ production in cells of monocytic origin or T cells and NK cells. Because of their ability to disrupt the production of IL 12 and TNF ⁇ in monocytes / macrophages and INF ⁇ production in T cells, the compounds of the present invention are useful in the treatment of many diseases caused by the increased production of cytokines, such as TNF ⁇ . ⁇ , IFN ⁇ , IL 2 and IL 12 are triggered, such as inflammatory diseases that are not based on neuroinflammation, autoimmune diseases, allergic and infectious diseases, toxin-induced inflammation, pharmacologically triggered inflammatory reactions and pathophysiologically relevant inflammatory reactions of currently unclear origin.
• cytokines such as TNF ⁇ . ⁇ , IFN ⁇ , IL 2 and IL 12 are triggered, such as inflammatory diseases that are not based on neuroinflammation, autoimmune diseases, allergic and infectious diseases, toxin-induced inflammation, pharmacologically triggered inflammatory reactions
• inflammatory and autoimmune diseases are: chronic inflammatory bowel disease (inflammatory bowel disease, Crohn's disease, ulcerative colitis), arthritis, allergic contact dermatitis, psoriasis, pemphigus, asthma, multiple sclerosis, diabetes, type I insulin-dependent diabetes mellitus, Rheumatoid arthritis, lupus and other collagenosis, Graves' disease, Hashimoto's disease, graft versus host disease and graft rejection.
• Examples of allergic, infectious and toxin-induced and ischemia-induced diseases are: Sarcoidosis, Asthma, Hypersensitive Pneumonitis, Sepsis, Septic shock, Endotoxin shock, Toxic Shock Syndrome, Toxic Liver Failure, ARDS (Acute Respiratory Syndrome), Eclampsia, Cachexia, Acute Viral Infections (eg mononucleosis, fulminant hepatitis), organ damage after reperfusion.
• An example of a pharmacologically triggered inflammation with pathophysiological relevance is the "first dose response" after administration of anti-
• T cell antibodies such as OKT3.
• neuroinflammatory diseases associated with microglial activation include AIDS dementia, amyotrophic lateral sclerosis, Creuzfeld-Jacob disease, Down 's syndrome, diffuse Lewy body disease, Huntington 's disease, leukencephalopathy, multiple sclerosis, Parkinson's disease , Pick's disease, Alzheimer's disease, stroke, temporary lobe epilepsy and tumors. Therefore, the invention also relates to the use of the specified benzimidazole derivatives for the treatment of these diseases and for the prophylaxis of these diseases.
• Microglia inhibitors useful in the present invention are compounds which, upon stimulation with the A ⁇ peptide, achieve at least 20% inhibition of microglial activity and at least 30% inhibition of cytokine activity.
• the biological properties of the microglial inhibitors can be demonstrated by the methods known to the person skilled in the art, for example with the aid of the investigation methods described below and in WO 01/51473.
• Example 29 describes how the inhibition of microglial activation can be measured.
• the microglia can be activated by various stimuli, such as A ⁇ -peptide [ ⁇ -amyloid, Araujo, DM and Cotman, CM., Brain Res., 569, 141-145 (1992)], with prion protein, cytokines or by cell fragments [Combs, CK. , et al., J. Neurosci., 19, 928-939, (1999), Wood, PL, Neuroinflammation: Mechanisms and Management, Humana Press, (1998)].
• Stimulation with the A ⁇ peptide corresponds to the pathophysiological situation in Alzheimer's disease.
• the substances according to the invention showed inhibition of microglial activation when stimulated with the A ⁇ peptide.
• the inhibition of microglial activation by the substances according to the invention leads to a strong reduction of cytokine production and secretion, for example of 111 ⁇ and TNF ⁇ (measured by ELISA and mRNA expression analysis), and to a reduced secretion of reactive oxygen / nitrogen intermediates , So there are several inhibited inflammatory factors.
• the in vivo efficacy of the substances of the invention was demonstrated in a MCAO model in rats. This model simulates the condition of a stroke.
• the substances according to the invention reduce the microglial activation that occurs in acute brain lesions in the brains of the animals.
• Inhibition of cytokine production is exemplified by measurement of TNF ⁇ and interleukin 12 in lipopolysaccharide (LPS) stimulated THP-1 cells.
• LPS lipopolysaccharide
• the compounds of the invention inhibit TNF ⁇ and interleukin 12 production in lipopolysanharide (LPS) stimulated THP-1 cells.
• LPS lipopolysanharide
• the compounds of the invention inhibit INF ⁇ production of peripheral blood mononuclear cells.
• the invention relates to pharmaceutical agents which contain one or more compounds of the general formula I according to the invention and one or more excipients.
• the pharmaceutical compositions or compositions of the invention are prepared with conventional solid or liquid carriers or diluents and customary pharmaceutical and technical excipients according to the desired mode of administration with a suitable dosage in a manner known per se.
• preferred Preparations consist of a dosage form suitable for oral, enteral or parenteral, for example, ip (intraperitoneal), iv (intravenous), im. (intramuscular), or percutaneous, application is appropriate.
• Such dosage forms are, for example, tablets, film-coated tablets, dragees, pills, capsules, powders, creams, ointments, lotions, liquids, such as syrups, gels, injectable liquids, for example for ip, iv, im., Or percutaneous injection, etc.
• depot forms are also contemplated , as implantable preparations, as well as suppositories suitable. Depending on their nature, the individual preparations give the derivatives according to the invention gradually or the entire amount to the body in a short time.
• capsules, pills, tablets, dragees and liquids or other known oral dosage forms may be used as pharmaceutical preparations.
• the drugs may be formulated to either release the drugs in a short time and deliver them to the body or have a depot effect, so that a longer-lasting, slow supply of active ingredient to the body is achieved.
• the dosage units may contain one or more pharmaceutically acceptable carriers, for example rheology-adjusting agents, surfactants, solubilizers, microcapsules, microparticles, granules, thinners, binders such as starch, sugar, sorbitol and gelatin Fillers, such as silica and talc, lubricants, dyes, fragrances and other substances.
• pharmaceutically acceptable carriers for example rheology-adjusting agents, surfactants, solubilizers, microcapsules, microparticles, granules, thinners, binders such as starch, sugar, sorbitol and gelatin Fillers, such as silica and talc, lubricants, dyes, fragrances and other substances.
• Corresponding tablets can be prepared, for example, by mixing the active compound with known excipients, for example inert diluents such as dextrose, sugar, sorbitol, mannitol, polyvinylpyrrolidone, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as carboxypolymethylene, carboxymethylcellulose, cellulose acetate phthalate or polyvinyl acetate , to be obtained.
• excipients for example inert diluents such as dextrose, sugar, sorbitol, mannitol, polyvinylpyrrolidone, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as carboxypolymethylene, carboxymethylcellulose, cellulose acetate phthalate or polyvinyl acetate , to be obtained.
• the tablets can also consist of several
• Coated tablets can accordingly be produced by coating cores produced analogously to the tablets with agents customarily used in tablet coatings, for example polyvinylpyrrolidone or shellac, gum arabic, talc, titanium oxide or sugar.
• the dragee wrapper can also consist of several layers, wherein the auxiliaries mentioned above in the case of the tablets can be used.
• Active ingredients containing capsules can be prepared, for example, by mixing the active ingredient with an inert carrier such as lactose or sorbitol and encapsulated in gelatin capsules.
• an inert carrier such as lactose or sorbitol
• the benzimidazole derivatives of the invention may also be formulated in the form of a solution intended for oral administration which, in addition to the active benzimidazole derivative, contains as ingredients a pharmaceutically acceptable oil and / or a pharmaceutically acceptable lipophilic surfactant and / or a pharmaceutically acceptable hydrophilic surfactant Substance and / or a pharmaceutically acceptable water-miscible solvent.
• the compounds can also be formulated as Cyclodextrinchlatrate.
• the compounds with ⁇ -, ß- or ⁇ -cyclodextrin or their derivatives are reacted.
• compositions according to the invention are supplied to the body in sufficient quantity.
• adjuvants are included, for example, rheology-adjusting agents, surfactants, preservatives, solubilizers, thinners, skin permeation enhancers for the benzimidazole derivatives of the present invention, dyes, perfumes and skin protection agents such as conditioners and moisturizers.
• the compounds according to the invention may also contain other active ingredients in the
• the substances according to the invention can also be used in suitable solutions, for example physiological saline, as an infusion or injection solution.
• suitable solutions for example physiological saline
• the active ingredients may be dissolved or suspended in a physiologically acceptable diluent.
• Particularly suitable diluents are oily solutions, for example solutions in sesame oil, castor oil and cottonseed oil.
• solubilizers such as benzyl benzoate or benzyl alcohol, may be added.
• any liquid carrier can be used in which the compounds according to the invention are dissolved or emulsified.
• These liquids often also contain substances for the regulation of viscosity, surface-active substances, preservatives, solubilizers, thinners and other additives with which the solution is rendered isotonic.
• Other active ingredients can also be administered together with the benzimidazole derivatives.
• the substances according to the invention are applied in the form of a depot injection or an implant preparation, for example subcutaneously.
• Such preparations may be formulated to allow sustained release of the active ingredient.
• known techniques can be used, for example, dissolving or working with a membrane depots.
• Implants may include as inert materials, for example, biodegradable polymers or synthetic silicones, for example silicone rubber.
• the benzimidazole derivatives may also be incorporated into a patch for percutaneous administration, for example.
• the dosage of the substances of the general formula I according to the invention is determined by the attending physician and depends inter alia on the substance administered, the route of administration, the disease to be treated and on the severity of the disease.
• the daily dose is not more than 1000 mg, preferably not more than 100 mg, which dose may be given as a single dose to be administered once or divided into two or more daily doses.
• the benzimidazoles of the formula I are accessible in different ways by processes known per se from the literature.
• N-aryl-2-nitrobenzenes (C) can be generated under various reaction conditions, for example by heating the reactants with or without a suitable inert solvent such.
• the amine used as a reactant can be used in excess as a solvent.
• the reactions are carried out both without and with bases (for example potassium carbonate, sodium hydride).
• bases for example potassium carbonate, sodium hydride.
• other adjuvants such.
• B. copper salts find use. Examples of the approach given here can be found in numerous papers, such as: D. Jerchel, H. Fischer, M. Graft, Ann. Chem., 575, 162 (1952) CAS, 53 (2138); RA. Abramovitch, Can. J. Chem., 38, 2273, 1960).
• the reduction of the nitro group (C) is preferably by hydrogenation in polar solvents such as acetic acid, lower alcohols or ethyl acetate with addition of catalysts such as Raney nickel or palladium on carbon, or by chemical reduction, for example with tin in hydrochloric acid, SnCl 2
• diamines (D) are by reaction with acid derivatives such as orthoesters, imino esters, acid anhydrides, aldehydes or free
• Carboxylic acids with or without acid catalysis and / or dehydrating agents the benzimidazoles (E) available.
• the preparation of 1, 2-diphenylbenzimidazole from benzoic acid and ⁇ / -phenyl-o-phenylenediamine using triphenylphosphine oxide and trifluoromethanesulfonic anhydride J. B. Hendrickson, M.S.
• aromatic aldehydes for example, nitrobenzene is used as the solvent in order to be able to carry out the oxidation of the benzimidazoline formed in situ in situ.
• nitrobenzene is used as the solvent in order to be able to carry out the oxidation of the benzimidazoline formed in situ in situ.
• the cyclization to benzimidazoles succeeds by using aromatic aldehydes as
• the substituents R must be compatible with the reagents and reaction conditions used in the course of the synthesis sequence.
• the substituents may be modified later. If the structural element BA-0 (formula I) is established in protected or unprotected form due to incompatibility with the reaction conditions during the respective Benzimidazolsynthese or other synthetic reasons only after completion of Benzimidazolsynthese, depending on the substituents R 3 brought along the benzene ring of benzimidazoles different approaches to Establishment of the .beta.-AO structural element (formula I) possible, which, of course, to the expert, a compatibility of the methods used with the aryl substituents and other radicals R 3 must be considered.
• solubilizers such as halogenated hydrocarbons or with boron tribromide in inert solvents such as dichloromethane
• the hydroxyl function can be implemented by known methods with optionally a terminal group B (formula I), or a precursor thereof containing alkyl halides to the ethers, the reaction with the alkylating agents preferably in polar solvents such as dimethylformamide, dimethyl sulfoxide, ethers, such as tetrahydrofuran or lower ketones, such as acetone or methyl ethyl ketone, with addition of bases, such as alkali metal and alkaline earth metal hydrides, but preferably sodium hydride, or with the addition of alkali metal carbonates, such as potassium or cesium carbonate, is carried out in a temperature range from 0 ° C to 120 ° C.
• polar solvents such as dimethylformamide, dimethyl sulfoxide, ethers, such as tetrahydrofuran or lower ketones, such as acetone or methyl ethyl ketone
• bases such as alkali metal and alkaline earth metal hydr
• a reaction in a two-phase system can be carried out under phase transfer catalysis, wherein the reactants are dissolved in a suitable inert organic solvent such as in Haloalkanes, but preferably in dichloromethane.
• the other phase is a solid alkali metal hydroxide, preferably sodium or potassium hydroxide, or else a concentrated aqueous solution of the hydroxide in question.
• phase transfer catalysts for example, quaternary ammonium salts are used. Reactions under phase transfer catalysis are preferably carried out at room temperature.
• the cleavage of the ester by acidic or alkaline hydrolysis can be carried out by the methods known in the art, such as with basic catalysts such as with alkali or alkaline earth metal carbonates or hydroxides in water or the aqueous solution of an alcohol.
• Suitable alcohols are aliphatic alcohols such as methanol, ethanol, butanol, etc., but preferably methanol.
• Aqueous solutions of ethers such as tetrahydrofuran are also used.
• alkali metal carbonates and hydroxides lithium, sodium and potassium salts may be mentioned. Preferred are the lithium and sodium salts.
• Suitable alkaline earth carbonates and hydroxides are, for example, calcium carbonate, calcium hydroxide and barium carbonate.
• the reaction is generally carried out at -10 ° C. to 70 ° C., but preferably at 25 ° C.
• the ester cleavage can also be carried out under acidic conditions, such as in aqueous hydrochloric acid, if appropriate with the aid of a solubilizer, such as a lower alcohol, preferably methanol, respectively.
• the alkylating reagents may also contain functional groups such as hydroxyl functions in free or protected form, which can be exchanged after conversion into leaving groups such as tosylate, mesylate, bromide or iodide, for example against amino or alkyl groups. Also, the alkylating reagents functional Groups, such as halogens or optionally protected amino groups, which can then be further modified.
• the substituents R 3 are contained in the synthesis building blocks from the outset or are established as required at a suitable site of the relevant synthesis sequence or generated from suitable precursors brought along.
• the free acid derivatives of the formula I or ester precursors can be converted into amide derivatives of the formula I by various processes known from the literature.
• the free acid derivatives of the formula I can also be converted into salts with suitable amounts of the corresponding inorganic bases with neutralization. For example, upon dissolution of the corresponding acids in water containing stoichiometric amounts of the base, after evaporation of the water or after addition of a water-miscible solvent, for example, alcohol or acetone, the solid salt is obtained.
• a water-miscible solvent for example, alcohol or acetone
• the amine salts can be prepared in the usual way.
• the corresponding acid is dissolved in a suitable solvent, such as, for example, ethanol, acetone, diethyl ether or benzene, and one to five equivalents of the respective amine of this solution are added.
• a suitable solvent such as, for example, ethanol, acetone, diethyl ether or benzene, and one to five equivalents of the respective amine of this solution are added.
• the salt usually accumulates in solid form or is isolated after evaporation of the solvent in a conventional manner.
• the clathrates with ⁇ -, ⁇ - or ⁇ -cyclodextrin are obtained analogously to the instructions in WO-A-87/05294.
• ⁇ -cyclodextrin is used.
• Liposomes are prepared according to the method described in Pharmacia in our time, __, 98 (1982). The preparation of some precursors, intermediates and products is described below by way of example. Unless the preparation of the starting compounds is described, the starting compounds are known and commercially available, or the compounds are synthesized analogously to the processes described.
• the compound to be reduced is dissolved in ethyl acetate, tetrahydrofuran, methanol or ethanol or mixtures of the solvents and hydrogenated at 2-5% (based on the nitro compound) of palladium on carbon (10%) at normal pressure. After the end of the hydrogen uptake is filtered off, the residue washed with ethyl acetate or methanol or ethanol and the filtrate was concentrated in vacuo. The crude product is usually reacted without further purification.
• a solution of 1.85 mmol of the hydroxybenzimidazole derivative in 12 ml of ⁇ /, ⁇ / -dimethylformamide is mixed with 1.85 mmol of cesium carbonate, and 2.24 mmol of alkylformamide. bromide or alkyl iodide added. When using the alkyl bromides optionally 1.85 mmol of sodium iodide are added. The mixture is stirred for 12-96 h, then poured into water, taken up in ethyl acetate, the organic phase is washed four times with water, dried over sodium sulfate and concentrated in vacuo. As an alternative to this aqueous work-up, the reaction mixture can be admixed with dichloromethane, separated from the precipitated salts by filtration and the filtrate concentrated in vacuo.
• the residue is purified by crystallization or column chromatography on silica gel.
• Example 14 5 - [[1- (4-Fluorophenyl) -2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] pentanoic acid a) 5 - [[1- (4-fluorophenyl) -2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] pentanoic acid methyl ester
• 6-Hydroxy-1-phenyl-2- (3-pyridinyl) -1H-benzimidazole was prepared by reacting 6-methoxy-1-phenyl-2- (3-pyridinyl) -1H-benzimidazole according to the general procedure 2 received.
• Example 16 4 - [[1-Phenyl-2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] butyric acid a) 4 - [[1-phenyl-2- (3-pyridinyl) -1 H- Benzimidazol-6-yl] oxy] butyric acid methyl ester was obtained by reacting 6-hydroxy-1-phenyl-2- (3-pyridinyl) -1H-benzimidazole with methyl 4-bromobutanoate according to general procedure 3. MS (El): 387 (molecular ion peak)
• Example 22 6 - [[1- (4-Methylphenyl) -2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] hexanamide was prepared by reacting 6 - [[1- (4-methylphenyl) -2 - (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] hexanoic acid methyl ester with ammonium chloride according to general procedure 6 shown.
• Example 23 ⁇ / -Cyclopropyl-6 - [[1- (4-methylphenyl) -2 - (- 3-thienyl) -1H-benzimidazol-6-yl] oxy] hexanamide was prepared by reacting 6 - [[1- (4-methylphenyl) -2- (3-thienyl) -1H-benzimidazol-6-yl] oxy] hexanoic acid methyl ester with cyclopropylamine according to general procedure 6 is shown. MS (El): 459 (molecular ion peak)
• Example 24 ⁇ / -Cyclopropyl-6 - [[1- (4-methylphenyl) -2 - (- 3-thienyl) -1H-benzimidazol-6-yl] oxy] hexanamide was prepared by reacting 6 - [[1- (4-methylphenyl) -2- (3-thienyl) -1H-benzimidazol-6
• 6-yl] oxy] pentanamide was prepared by reacting methyl 5 - [[1- (4-methylphenyl) -2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] pentanoate with 2-methoxyethylamine in accordance with general working instruction 6 shown.
• Example 27 5 - [[1- (4-Methylphenyl) -2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] pentanamide was prepared by reaction of 5 - [[1- (4-methylphenyl) -] 2- (3-pyridinyl) -1H-benzimidazol-6-yl] oxy] pentanoic acid methyl ester with ammonium chloride according to general procedure 6 shown.
• a ⁇ -activated microglia For in vitro production of A ⁇ -activated microglia, primary rat microglia are incubated with synthetic A ⁇ peptide: To simulate A ⁇ deposits, synthetic A ⁇ peptide is dried on 96-well tissue culture plates. For this purpose, a peptide stock solution of 2 mg / ml H 2 O 1: 50 in H 2 0 diluted. To coat the 96-well plates, 30 ⁇ L of this diluted peptide solution / hole are used and dried overnight at room temperature.
• Primary rat microglia are harvested from mixed glial cultures obtained from P3 rat brains. To produce mixed glial cultures, the brains are removed from 3-day-old rats and freed of meninges.
• the cell separation is achieved by trypsinization (0.25% trypsin solution, 15 min 37 ° C)). After removal of un-digested Tissue fragments with the aid of a 40 ⁇ m nylon mesh are centrifuged off the isolated cells (800 rpm / 10 min). The cell pellet is resuspended in culture medium and transferred to 100 ml tissue culture flasks. (1 brain / tissue culture flask).
• DMEM Dulbecco's modified Eagle medium
• FCS fetal Calf serum
• ⁇ 10 4 microglia / well are seeded on A ⁇ -coated tissue culture plates and incubated for 7 days in DMEM (with glutamine) supplemented with penicillin (50 U / ml), streptomycin ( 40 ⁇ g / ml) and 10% (v / v) fetal calf serum (FCS) at 37 ° C and 5% C0 2 .
• DMEM with glutamine
• penicillin 50 U / ml
• streptomycin 40 ⁇ g / ml
• 10% (v / v) fetal calf serum (FCS) at 37 ° C and 5% C0 2 .
• FCS fetal calf serum
• the metabolic activity is via reduction of MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (sulfophenyl) -2H-tetrazolium ), Owen's Reagent, Baltrop, JA et al. Bioorg. & Med. Chem. Lett. 1, 6111 (1991)).
• the percent inhibition refers to a control treated with DMSO only.
• the compounds of the invention inhibit microglial activation
• the compounds of the invention were tested for in vivo activity in an animal model of cerebral ischemia (stroke), the MCAO (permanent middle cerebral artery occlusion) model.
• stroke cerebral ischemia
• MCAO permanent middle cerebral artery occlusion
• a cerebral infarction is triggered, which is due to the undersupply of the corresponding brain area with oxygen and Nutrients. Consequence of this undersupply is a pronounced
• microglial activation reaches its maximum only after several days and can last for several weeks.
• the compounds according to the invention were administered intraperitoneally 1-6 days after occlusion. The animals were perfused on day 7 and killed. The extent of microglial activation was measured by a modified immunohistochemical method. For this purpose, vibratome sections of fixed brains were incubated with antibodies that recognize the CR3 complement receptor or the MHCII complex on activated microglia. The quantification of the primary antibody binding was carried out by an enzyme-linked detection system. Treatment with the compounds according to the invention led to a reduction in microglial activation in the brain hemisphere affected by cerebral infarction.
• Inhibition of cytokine production is exemplified by measurement of TNF ⁇ and interleukin 12 in lipopolysaccharide (LPS) stimulated THP-1 cells.
• LPS lipopolysaccharide
• THP-1 cells (American Type Culture Company, Rockville, Md) / mL RPMI 1640 Medium (Life technologies) / 10% FCS (Life Technologies, Cat # 10270-106) to 96 wells.
• Shallow soil cell culture plates (TPP, product no. 9296) seeded (100 ⁇ L / well)
• the compounds according to the invention are added in different concentrations and preincubated for 30 minutes. The predilution of the test substances was carried out in incubation medium.
• the test substances are added as a 2-fold concentrated substance solution (100 ⁇ l (hole).)
• the cells were stimulated overnight at 37 ° C. with 0.1 ⁇ g / ml LPS (Sigma L2630, from E. coli serotype 0111.
• TNF ⁇ or the amount of interleukin 12 was determined quantitatively.To measure TNF.alpha., A commercially available TNF.alpha. Kit from CIS bio international was used (Product No. 62TNFPEB).
• the interleukin 12 amount was determined using the ORIGEN technology (IGEN International, Inc, Gaithersburg, Maryland) performed The calculated IC 5 o value corresponds to the concentration of test substance which is required strength to cause a 50% inhibition of the maximum TNFa or interleukin 12 production to achieve.
• the compounds of the invention inhibit TNF ⁇ and interleukin 12 production in lipopolysaccharide (LPS) stimulated THP-1 cells.
• LPS lipopolysaccharide
• the measurement of INF ⁇ secretion is used.
• the upper plasma layer is pipetted off and transferred to underlying cell layer (peripheral blood mononuclear cells) in 15 ml sample tubes (Falcon) and then several times with 10 ml HBSS HANKS balanced Solution (without Mg 2+ and Ca 2+ ), Cat. No. 14175-53 ) washed. Finally, the cell pellet is incubated in culture medium RPMI 1640 +25 mM Hepes (Life Technology Cat. No. 52400-04110% FCS (Life Technologies, cat. No. 10270-106), 0.4% penicillin-streptomycin solution (Life Technologies, cat . no. 15140- 106) resuspended (1 x 10 6 cells / ml).
• TNF ⁇ and IL-12 HD p70 production are demonstrated, for example, by measuring TNF ⁇ and IL-12 HD p70 in lipopolysaccharide (LPS) and interferon gamma (IFN ⁇ ) stimulated peripheral blood mononuclear cells.
• LPS lipopolysaccharide
• IFN ⁇ interferon gamma
• whole human blood was used (blood collection via Na citrate S-Monovetten "Coagulation 9 NC / 10 ml Sarstedt).
• Enrichment of the lymphocytes and monocytes was carried out by density gradient centrifugation. 15 ml of Histopaque-1077 (Sigma, cat. No. H8880) were introduced into 50 ml of LEUCOSEP tubes (Greiner, cat. No.
• the substances according to the invention were added in different concentrations as a 3-fold concentrated substance solution (100 ⁇ l / well).
• the stimulation of the cells was carried out at 37 ° C and 5% C0 2 over a period of 24 h. Thereafter, the cell culture supernatant was harvested and the concentrations of TNF ⁇ and IL-12 HD p70 were determined by means of commercially available ELISA kits from BioSource International (TNF- ⁇ EASIA, Cat. No. KAC1752) and R & D Systems (Quantikine TM HS IL-12, Cat No. HS 120).
• the calculated IC 5 o value corresponds to the concentration of test substance which is required to cause a 50% inhibition to achieve 12 HD p70 production of the maximum TNF ⁇ - or interleukin.
• the compounds of the present invention inhibit TNF ⁇ and IL-12 HD p70 production of peripheral blood mononuclear cells.
• Induction of IL-10 Production of Peripheral Blood Mononuclear Cells Induction of IL-10 production is exemplified by measurement of IL-10 in phytohemagglutinin (PHA) or lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells.
• PHA phytohemagglutinin
• LPS lipopolysaccharide
• peripheral blood mononuclear cells whole human blood was used (blood collection via Na citrate S-Monovetten "Coagulation 9 NC / 10 ml Sarstedt). Enrichment of the lymphocytes and monocytes was carried out by density gradient centrifugation. 15 ml of Histopaque-1077 (Sigma, cat. No. H8880) were introduced into 50 ml of LEUCOSEP tubes (Greiner, cat. No. 227290) and centrifuged for 30 seconds at 250g pushed down through the frit in the tube. Subsequently, 20 ml of whole blood are added and centrifuged for 15 min. At 800 g and room temperature.
• the supernatant (plasma and platelets) is pipetted off and discarded and the underlying cell layer (lymphocytes and monocytes) in 50 ml Centrifuge tube (Falcon) and then washed 3 times in culture medium VLE RPMI 1640 (Seromed, No. FG1415) (centrifugation 10 min. At 250g, room temperature). Finally, the cell pellet is incubated in culture medium VLE RPMI 1640 (Seromed, No. FG1415), 10% FCS (Life Technologies, Cat. No.
• the substances according to the invention were added in different concentrations as a 3-fold concentrated substance solution (100 ⁇ l / well).
• the stimulation of the cells was carried out at 37 ° C and 5% C0 2 over a period of 24 h. Thereafter, the cell culture supernatant was harvested and IL-10 quantified.
• the IL-10 concentration was determined using a commercially available ELISA kit from BioSource International (Human IL-10, cat.No .: KHC0101C).
• the calculated EC 50 value corresponds to the concentration of test substance needed to increase IL-10 secretion by 50% of the maximum increase.
• the compounds of the invention increase the IL-10 production of peripheral blood mononuclear cells.

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